The diagnosis of diseases through analysis of human breath has long been practiced in medicine. For example, by smell alone, various volatile components of breath such as acetone, ammonia or sulfur compounds can be detected and provide information used to evaluate conditions such as diabetes, liver impairment and kidney disfunction. Gas chromatography and mass spectrometry also have been applied to evaluate exposure to toxic substances, liver disease and lung cancer.
Thus, the measurement of exhaled substances may be useful as a diagnostic and prognostic tool in a variety of medical conditions for a wide variety of medical conditions. Often, it is of interest when assessing pulmonary function to measure one or more of a variety of exhaled substances. These include endogenous gases (ie., oxygen, carbon dioxide and nitric oxide), exogenous gases used to test pulmonary diffusing capacity (ie., carbon monoxide, acetylene, argon and helium), volatile substances (i.e., ethane and pentane) and non-volatile substances (i.e., proteins such as surfactants, DNA and hydrogen peroxide) often found by sampling the liquid present in exhaled breath (i.e., breath condensate).
For example, the detection of several non-volatile macromolecules in exhaled breath has been evaluated as a possible diagnostic tool. However, identical molecules may also arise in the nasal passages. See, generally, Scheideler et al., Am. Rev. Respir. Dis. 148:778-784 (1993). Thus, proteins in breath condensate have been collected and separated by two-dimensional polyacrylamide gel electrophoresis. Such samples were analyzed by immunoassay for inflammation related proteins such as interleukin-1, interleukin-2, tumor necrosis factor .alpha., and others. Id. The level of leukotriene B4, a mediator of mucosal inflammation, was found to be elevated in the breathing condensate of patients with bronchopulmonary disease. Becher et al., App. Cardiopulmonary Path. 5:215-219 (1995). Similarly, various compounds have been found to be elevated in patients with bronchogenic carcinoma. See, e.g., U.S. Pat. No. 4,772,559 to Preti et al. Also, the detection of pathogenic microorganism DNA in the airways has been evaluated by detecting isolated DNA in human exhalate. Hillebrand et al, ATS Abstracts (1996):181.
As another example of the importance of monitoring the components of exhaled breath, patients with stable and unstable chronic obstructive pulmonary disease exhibit increased oxidant production in the airways, increasing further during exacerbations, and levels can be monitored by measuring exhaled hydrogen peroxide. See, e.g., Dekhuijzen et al, M. J. Resp. & Crit. Care Med. 154:813-816 (1996). Thus, the measurement of exhaled hydrogen peroxide is a marker fox acute airway inflammation in pediatric asthma patients. Dohlman et al, M. Rev. Resp. Disease 148:955-960 (1993).
One exhaled substance of particular interest is exhaled endogenous nitric oxide (NO). Nitric oxide is now known to be a central mediator in biological systems and, therefore, endogenous exhaled nitric oxide is thus potentially of interest in the diagnosis and monitoring of pulmonary function and various pulmonary diseases. Nitric oxide can be measured in the exhaled breath of animal and human subjects and shows particular promise as a diagnostic tool useful in evaluating inflammatory airway diseases, in particular bronchial asthma, and also in evaluating bronchiectasis and lung transplant rejection and other pulmonary conditions. A recent article coauthored by the present inventors summarizes published values and techniques for measuring exhaled nitric oxide. See, Silkoff et al., Am J. Resp. Crit. Care Med. 155:260-267 (1997) and the references cited therein as well as Table 1, below.
For example, asthmatic patients have relatively high exhaled NO levels as compared to normal subjects and these levels decrease rapidly after the institution of anti-inflammatory therapy. See, e.g., Kharitonov, et al., Lancet 343:133-135 (1994). Thus, measuring exhaled NO in conjunction with existing tests may aid in the diagnosis and assessment of asthma, and also be an index of the response to therapy, or patient compliance in therapy. In view of the importance of asthma as a major health problem, the commercial potential is great for tests that can help diagnose assess severity and ascertain the response to therapy.
A variety of systems have been developed to collect and monitor exhaled breath components, particularly gases. For example, U.S. Pat. No. 3,951,607 to Fraser describes a gas analyzer for pulmonary use that is connected to appropriate detectors for, e.g., nitrogen, oxygen, carbon dioxide, carbon monoxide, helium, acetylene, nitrous oxide, nitric oxide, sulphur dioxide and anesthetic gases. Various other apparatus for collecting and analyzing expired breath include the breath sampler of Glaser et al, U.S. Pat. No. 5,081,871; the apparatus of Kenny et al, U.S. Pat. No. 5,042,501; the apparatus for measuring expired breath of infants of Osborn, U.S. Pat. No. 4,202,352; and the instrument for parallel analysis of metabolites in human urine and expired air of Mitsui et al., U.S. Pat. No. 4,734,777. Pulnonary diagnostic systems including computerized data analysis components also are known, e.g., Snow et al., U.S Pat. No. 4,796,639. Some detection systems rely upon mass spectrographic equipment and others rely upon rapid-response chemiluminescent analyzers such as Sievers Instruments, Inc. (Boulder, Colo.) Model 270B, which is preferred for the measurement of exhaled nitric oxide.
Notwithstanding the various known breath collection and analysis systems, published methods to date may be confounded by two problems. First, in order to measure the amount of substances originating from the lower respiratory tract as opposed to the upper respiratory tract (i.e., the paranasal sinuses and nasal cavities), a more informative system must substantially eliminate or exclude such substances to the extent that they originate from the upper respiratory tract, i.e., above the velum (or soft palate). For example, nitric oxide emerging from the nasal cavity is present in high concentrations relative to the level of nitric oxide originating in the lower respiratory tract, often in the parts per million range, and thus is present at levels that are an order of magnitude greater than those in the airways below the glottis. Such nasal cavity nitric oxide enters the airstream via the nasopharynx and then emerges through the mouth, and it preferably should be excluded. The present inventors have found that apparatus utilizing, e.g., a nose clip and low resistance mouthpiece, such as are used to monitor exhaled gases during exercise, are not adequate to satisfy the foregoing concern. Such a system is described, e.g., by Morrison et al., Am. J. Cardiol. 64:1180-1184 (1989).
Second, when measuring exhaled NO, for example, concentrations are altered (i.e., almost 35-fold) greatly by the expiratory flow rate, likely by affecting the transit time in the airway. The expiratory flow rate changes the transit time in the airway and thus changes the time available for NO uptake. Moreover, different people breath at different rates. Thus, a means for providing even and consistent flow rates also are important.
What has been needed, therefore, is a technique and associated equipment for receiving, collecting and sampling the components of exhaled breath in which contamination with substances present or originating in the upper respiratory tract, e.g., the nasal cavity, such as those originating from the nasal mucosa, is prevented or substantially reduced. Additionally, because an uncontrolled expiratory flow rate may complicate the measurement and evaluation of samples, techniques and methods to compensate for and substantially reduce variability also have been needed. The present invention thus is directed to such techniques and to associated equipment. Methods according to the invention are eminently suitable for both the inpatient and outpatient setting. The disclosed methods are reproducible, quick and easy to perform by medical staff and comfortable for the subject so that a pulmonary exhaled breath measurement system could become a routine part of the lung function assessment in every respirology clinic.